In mass spectrometry sample molecules are ionized and then the ions are analyzed to determine their mass-to-charge (m/z) ratios. The ions can be produced by a variety of ionization techniques, including electron impact, fast atom bombardment, electrospray ionization (ESI) and matrix-assisted laser desorption ionization (MALDI). The analysis by m/z is performed in analyzers in which the ions are either trapped for a period of time or fly through towards the ion detector. In the ion trapping analyzers, such as radiofrequency quadrupole ion trap (Paul trap), linear ion trap and ion cyclotron resonance (ICR) analyzers (Penning trap), the ions are spatially confined by a combination of magnetic, electrostatic or alternating electromagnetic fields for a period of time typically from about 0.1 to 10 seconds. In the transient-type mass analyzers, such as magnetic sector, quadrupole, and time-of-flight analyzers, the residence time of ions is shorter, in the range of about 1 to 100 μs.
Tandem mass spectrometry is a general term for mass spectrometric techniques where sample ions (precursor ions) of desired m/z values are selected and dissociated inside the mass spectrometer and the obtained fragment ions are analyzed according to their m/z values. Dissociation of mass-selected ions can be performed in a special cell between two m/z analyzers. This cell is usually a multipole ion trap, i.e. quadrupole, hexapole, etc. ion trapping device. In ion trap mass spectrometry instruments, the dissociation occurs inside the trap (cell). Tandem mass spectrometry can provide much more structural information on the sample molecules.
Tandem mass spectrometry is a general term for mass spectrometric techniques, where sample ions (precursor ions) of desired m/z values are selected and dissociated inside the mass spectrometer once (MS/MS or MS2) or multiple times (n-times: MSn) before the final mass analysis takes place.
To fragment the ions in the mass spectrometer, collision-induced dissociation (CID) or infrared multiphoton dissociation (IRMPD) are most commonly employed. Both of these techniques produce vibrational excitation (VE) of precursor ions above their threshold for dissociation. In collision-induced dissociation, VE is achieved when precursor ions collide with gas atoms or molecules, such as e.g. helium, argon or nitrogen, with subsequent conversion of the collisional energy into internal (vibrational) energy of the ions. Alternatively, the internal energy may be increased by sequential absorption of multiple infrared (IR) photons when the precursor ions are irradiated with an IR laser. These precursor ions with high internal energy undergo subsequent dissociation into fragments (infrared multiphoton dissociation, IRMPD), one or more of which carry electric charge. The mass and the abundance of the fragment ions of a given kind provide information that can be used to characterize the molecular structure of the sample of interest.
All VE techniques have serious drawbacks. Firstly, low-energy channels of fragmentation always dominate, which can limit the variety of cleaved bonds and thus reduce the information obtained from fragmentation The presence of easily detachable groups results in the loss of information on their location. Finally, both collisional and infrared dissociations become ineffective for large molecular masses.
To overcome these problems, a number of ion-electron dissociation reactions have been proposed (see the review by Zubarev, Mass Spectrom. Rev. (2003) 22:57–77). One such reaction is electron capture dissociation (ECD) (see Zubarev, Kelleher and McLafferty J. Am. Chem. Soc. 1998, 120, 3265–3266). In the ECD technique, positive multiply-charged ions dissociate upon capture of low-energy (<1 eV) electrons produced either by a heated filament, or by a dispenser cathode as in Zubarev et al. Anal. Chem. 2001, 73, 2998–3005. Electron capture can produce more structurally important cleavages than collisional and infrared multiphoton dissociations. In polypeptides, for which mass spectrometry analysis is widely used, electron capture cleaves the N—Cα backbone bonds, while collisional and infrared multiphoton excitation cleaves the amide C—N backbone bonds (peptide bonds). Moreover, disulfide bonds inside the peptides, that usually remain intact in collisional and infrared multiphoton excitations, fragment specifically upon electron capture. Finally, some easily detachable groups remain attached to the fragments upon electron capture dissociation, which allows the determination of their positions. This feature is especially important in the analysis of post-translational modifications in proteins and peptides, such as phosphorylation, glycosylation, γ-carboxylation, etc. as the position and the identity of the post translationally attached groups are directly related to the biological function of the corresponding peptides and proteins in the organism.
Other ion-electron fragmentation reactions also provide analytical benefits. Increasing the electron energy to 3–13 eV leads to hot-electron capture dissociation (HECD), in which electron excitation precedes electron capture. The resulting fragment ions undergo secondary fragmentation, which allows to distinguish between the isomeric leucine and isoleucine residues (see Kjeldsen, Budnik, Haselmann, Jensen, Zubarev, Chem. Phys. Lett. 2002, 356, 201–206). In electron detachment dissociation (EDD) introduced by Budnik, Haselmann and Zubarev (Chem. Phys. Lett. 2001, 342, 299–302), 20 eV electrons ionize peptide di-anions, which produces effect similar to ECD. EDD is advantageous for acidic peptides and peptides with acidic modifications, such as sulfation.
In order to make the bookkeeping of the hydrogen atom transfer to and from the fragments easier, the “prime” and “dot” notation has been introduced. In this notation the presence of an unpaired electron is always noted with a radical sign “.”, e.g. homolytic N—Cα bond cleavage gives c. and z. fragments. Hydrogen atom transfer to the fragment is denoted by a “′”, e.g. hydrogen transfer to c. gives c′ species, while hydrogen atom loss from z. results in z′ fragments.
Combined use of ion-electron fragmentation reactions with VE techniques provides additional sequence information (see Horn, Zubarev and McLafferty, Proc. Natl. Acad. Sci. USA, 2000, 97, 10313–10317). First, ion-electron reactions produce not only more abundant, but also different kind of cleavage (e.g. N—Cα bond cleavage giving c′n and z.n ions) than VE techniques (C—N bond cleavage yielding bn and y′n ions). Comparison between the two types of the cleavage allows one to determine the type of the fragments. For example, the mass difference between the N-terminal c′n and bn ions is 17 Da, while that between the C-terminal y′ and z. ions is 16 Da. Second, the cleavage sites are often complementary. For instance, VE techniques cleave preferentially at the N-terminal side of the proline residues, while this site is immune to ECD. On the other hand, ECD cleaves S—S bonds preferentially, while these bonds remain intact in most VE experiments. Finally, polypeptides with post-translational modifications exhibit in VE characteristic losses, which allows one to identify the presence and type of the modification. At the same time, ECD affords determination of the sites of modifications (see Kjeldsen, Haselmann, Budnik, Sørensen and Zubarev, R. A. Anal. Chem. (2003), 2003, 75:2355–2361). Although ion-electron reactions can be used simultaneously with VE techniques, the complementary character of the analytical information obtained in these techniques favors independent consecutive use of them (Tsybin, Witt, Baykut, Kjeldsen, and H{dot over (a)}kansson, Rapid. Commun. Mass Spectrom. 2003, 17, 1759–1768).
A drawback of current tandem mass spectrometry utilizing both ion-electron reactions and VE techniques is that the consecutive use of these reactions demands at least twice as much time for the analysis as is required by the fastest of these techniques. This time of the analysis is especially critical while analyzing low-concentration samples, which is the case in biological mass spectrometry where the sample quantity is often limited. Low-concentration samples require either long (several seconds) accumulation of the precursor ions in the trapping device, or integration of many individual MS/MS spectra. In both cases, the time loss due to the consecutive use of ion-electron reactions and VE techniques can be in the order of several seconds. This severely limits the analytical utility of tandem mass spectrometry when it is combined with the separation techniques, such as liquid chromatography (HPLC) or capillary electrophoresis (CE), where the entire signal from an individual compound often lasts for just a short period of time not exceeding some seconds. Therefore, while separating or simultaneously using VE and ion-electron reactions on-line with both HPLC and CE has been demonstrated, consecutive use of these fragmentation techniques on-line with separation techniques, although deemed highly advantageous in e.g. Kjeldsen, Haselmann, Budnik, Sørensen and Zubarev, Anal. Chem. 2003, 75, 2355–2361, has not been achieved yet because of the time-of-analysis limitations.